BIOETHANOL PRODUCTION FROM PRETREATED SUGARCANE BAGASSE UNDER OPTIMISED CONDITIONS USING SELECTED FUNGI

Abstract

Sugarcane Bagasse (SB) is a major waste of the sugar industry and constitutes disposal problem in the environment. The bagasse is known to contain cellulose and hemicellulose which can be converted to bioethanol. However, the recalcitrant nature of plant biomass demands optimal pretreatment method to make sugar components available for enzymatic depolymerisation. Therefore, this study was designed to optimally pretreat SB and to identify appropriate fungi for enhanced bioethanol yield. Fungi (moulds and yeasts) were isolated from SB collected from a sugar industry dumpsite using pour-plate method. Standard methods were used to screen organisms (105 CFU/ml) for their ability to produce cellulases and hemicellulases. Selected isolates were identified using molecular techniques. Yeasts were further screened based on their ability to convert pentose and hexose sugars to bioethanol using different nitrogen sources to select the appropriate yeast. Yeast tolerance to temperature, acetic acid, ethanol and furfural was determined using turbidimetry. Optimisation of pretreatment of SB at different concentrations of potassium hydroxide (KOH), temperature and treatment time was determined using Response Surface Methodology (RSM). Pretreated SB was hydrolysed using selected moulds, while a commercial hemicellulase mixture served as control. Fermentation of pretreated SB hydrolysate with selected yeasts using Separate Hydrolysis and Fermentation (SHF) as well as Simultaneous Saccharification and Fermentation (SSF) of pretreated SB were also carried out. Bioethanol yield was determined; and data were subjected to descriptive statistics. A total of 120 yeasts and 21 moulds were isolated. Aspergillus niger XY was the highest enzyme producer for endoglucanase (60.34±0.72 U/ml), beta-glucosidase (14.29±0.02 U/ml) and xylanase (82.67±0.65 U/ml). Eleven yeasts grew on both glucose and xylose and were identified as Pichia kudriavzevii (7), Saccharomyces cerevisiae (1), and Candida tropicalis (3). All yeasts converted glucose to ethanol but only C. tropicalis Y5 converted xylose to ethanol (4.83 g/l) with urea as the best nitrogen source. Pichia kudriavzevii Y2, C. tropicalis Y5 and S. cerevisiae Y10 tolerated temperatures up to 48 oC and 17.5% ethanol. Pichia kudriavzevii Y2 and S. cerevisiae Y10 adapted up to 6 g/l acetic acid with 49% and 45% growth while C. tropicalis Y5 adapted to 7 g/l acetic acid with 34% growth iii after 48 hours of incubation. The isolates were able to adapt to 3 g/l furfural concentration with percentage growth of 53%, 47% and 46% for P. kudriavzevii Y2, C. tropicalis Y5 and S. cerevisiae Y10, respectively. Optimum pretreatment conditions were: 150 mg/g bagasse (KOH), 86 oC and 120 minutes. Hydrolysis with hemicellulase yielded reducing sugars of 600 mg/g bagasse within 20 hours while hydrolysis with A. niger XY took a longer time (12 days) and yielded 18.8 mg/g bagasse. Bioethanol yield using SHF and SSF were 19 g/l and 30 g/l, respectively. Alkaline pretreatment followed by enzymatic hydrolysis gave a higher yield of total reducing sugars. Candida tropicalis Y5 converted both pentose and hexose to bioethanol and showed good prospect for its use in commercial fermentation of sugarcane bagasse.